Abstract Endothelium-derived relaxing factor, identified as nitric oxide (NO), is a key mediator regulating vascular tone and blood pressure. NO mediates vascular relaxation through activation of soluble guanylate cyclase (sGC) in the smooth muscle. While NO is synthesized by a specific well characterized NO synthase in the endothelium (eNOS), the process of vascular NO degradation and metabolism is poorly understood. It is hypothesized that NO degradation in the vessel wall is mediated by an O2-dependent NO dioxygenase (NOD) that oxidizes NO to nitrate. However, the identity of the specific NO dioxygenase that serves as the main in vivo regulator of O2- dependent NO degradation in vascular smooth muscle has remained elusive. Cytoglobin (Cgb) is a recently discovered globin expressed in smooth muscle with unknown function. Based on our preliminary data where we observe that: 1) Cgb is the major globin expressed in smooth muscle; and 2) knockout of Cgb greatly prolongs NO decay, increases vascular relaxation, lowers blood pressure and systemic vascular resistance, we hypothesize that Cgb is the major heme protein regulating the rate of O2-dependent NO metabolism in the vessel wall, in turn profoundly modulating vascular tone. In this grant, there are 3 aims in which we sequentially test this hypothesis, first in vascular smooth muscle cells, then in isolated vessels and finally in the in vivo cardiovascular system with experimental measurements and computational modeling. The latter will enable us to determine how well the process of Cgb-mediated O2-dependent NO metabolism accounts for the measured vascular NO metabolism along with enabling prediction of the effects of modulating Cgb expression and NOD function. Since the O2-dependent NOD function of Cgb is controlled by its rate of reduction, studies will be performed to elucidate the process and mechanisms of Cgb reduction in smooth muscle cells, vessels and in vivo in mice utilizing molecular knockdown, pharmacological inhibition, or other manipulation of these reducing pathways. We will determine how modulation of the NOD function of Cgb modulates vascular tone in normal physiology and vascular disease with studies in vessels and mice with varying Cgb expression levels and inhibition of each of the major pathways of its reduction or specific inhibition of its NOD function. These studies will be performed in normal mice and mice with angiotensin-induced hypertension. In these settings, studies will be performed to measure and scavenge superoxide (O2-.) to determine how the O2-dependent reaction of NO with Cgb compares to that of O2-. and specific attention paid to the role of the endothelium and the coupling state of eNOS in NO metabolism. Accomplishment of this research plan will elucidate the molecular mechanism underlying the important role of Cgb in vascular NO metabolism and provide important insights into the regulation of vascular tone in normal physiology and cardiovascular disease. This work will illuminate the path leading to development of novel therapeutic approaches to reverse vascular dysfunction and ameliorate secondary disease through modulation of the process of NO degradation.